Benzodiazepines, GABAergic drugs and the GABA-A receptor

Benzodiazepines are GABA agonists, binding indirectly to the GABA-A receptor but not the ligand binding site itself, and GABA binding to the ligand site is still neccessary for ion channel opening. Benzodiazepines bind to an allosteric site on the receptor know as the benzodiazepine binding site. When benzodiazepines are bound to the receptor in the presence of GABA, ion pore opening is enhanced, and therefore allowing greater conductance of Cl- ions through the pore.

The GABA-A receptor is a heteromer consisting of 5 protein subunits (pentamer) in a ring shape around a central pore. All GABA-A receptors contain α and β subunits, with most containing 2α and 2β subunits and 1γ subunit (α₂β₂γ). 6 different α _{subunits, 3}β and ₃γ subunits exist, so there are many possibilities. The GABA binding site is located between alpha and beta subunits, making two per receptor protein, and the benzodiazepine binding site exists between the alpha and gamma subunits, making one available in a common receptor protein. Note: Not all GABA-A receptors have a benzodiazepine site - some do not have gamma subunits, and α4 and α6 subunits are arginine not histine based meaning they cannot functionally bind with benzodiazepines. Binding sites with an α1 subunit  may higher affinity for more sedative benzodiazepines over binding sites with α2 or α3 subunits, which may be associated with drugs with a more anxiolytic effect.

Benzodiazepines - A Drug History

Benzodiazepines are a class of drugs with varying sedative, hypnotic, anxiolitic, anticonvulsant and muscle relaxant properties based on a core molecule containing a benzene and diazepine ring. The first benzodiazepine (chlordiazepoxide) was discovered in 1955 and the chlordiazepoxide and diazepam were released by Hoffmann LaRoche in the 60's with the brandnames Librium and Valium respectively. They changed the world of pharmacology and medical prescribing (along with meprobamate and methaqualone) by replacing barbiturates for most uses, and in time replacing methaqualone and its derivatives too with one major reason being the therapeutic index for clinical benzodiazepine use is far wider than many drugs with similar properties with death rarely occuring from overdose of a benzodiazepine if taken alone.

Benzodiazepines were considered relatively safe and problem-free at least compared to the drugs they replaced. They quickly became prescribed for a variety of uses. The Rolling Stones released a song called "Mothers Little Helper" in 1967 about Valium, with the lyrics:-  

"She goes running for the shelter

Of a mother's little helper

And it helps her on her way

Gets her through her busy day."

Valium became the biggest pharmaceutical seller in the US for the 1970's with 2.3bn tablets prescribed in 1978. The drugs were directly marketed to the public as well as to doctors. During the 70s, 1 in 5 European women and 1 in 10 European men would be prescribed a benzodiazepine at some point annually. As the 70s progressed reports of tolerance, dependence and withdrawal symptoms began to increase. In the 1980s media interest grew as evidence reguarding the use of benzodiazepines became more and more convincing. The largest ever British class-action lawsuit concerned drug manufacturers involving over 14,000 plaintiffs - an event that changed procedures for class action litigation in the UK.
Benzodiazepine prescribing was clamped down upon in most countries because of widespread use and misuse. Although all drugs of the class have misuse and abuse potential, some have gained notorioty. Flunitrazepam (Rohypnol) is synonomous for the 'Date Rape' drug even if its use as a date rape drug is rare in many countries. Temazepam has a long history of misuse in Europe, particularly Scotland and particularly when available in liquid filled capsules, the drug was injected. Sometimes benzodiazepines are still injected but because of low solubility necrosis may occur. Benzodiazepines in combination with opiates or alcohol are a the most common cause of accidental overdose and benzodiazepine-alcohol combinations are common in deliberate overdoses.
Modern day prescribing of benzodiazepines is very much different to the "cure for all ailments" attitude of the 60's and 70's - They are however of great clinical use for a variety of reasons such as:-

• Status epilepticus
• Short-term treatment of acute anxiety
• Short-term treatment of acute insomnia
• Alcohol and opiate withdrawal
• Pre/Post-operative sedation 

According to the British National Formulary:- 

“Benzodiazepines are indicated for the short-termrelief (2-4
weeks only) of anxiety that is severe, disabling or subjecting
the individual to unacceptable distress, occurring alone or in
association with insomnia or short-term psychosomatic,
organic or psychotic illness.”

Indeed, despite the risks in prescribing benzodiazepines the clinical benefits to patients may outway those risks:-

"…Benzodiazepines will usually be reserved for the
treatment of patients who have not responded to at least
two treatments (such as after non-response to both an
SSRI and a psychological treatment) but concerns about
potential problems in long term use should not prevent their
use in patients with persistent, severe, distressing, and
impairing anxiety symptoms (Nutt, 2005)."

GABA receptors

Gamma-amino butyric acid - Skeletal structure

GABA is the primary inhibitory neurotransmitter in the brain. Crudely, GABA is the function opposite of glutamate, the primary excitatory neurotransmitter in the brain. They have a close interrelationship with GABA even synthesized from glutamate (as a precursor) by glutamate decarboxylase (GAD).

There are two superfamilies of GABA receptor, GABA-A receptors and GABA-B receptors:-

GABA-A receptor
The GABA(A) receptor is an ionotropic ligand gated channel. Of the two families of receptor, it is by far the most understood and is a target of a huge number of drugs including those for anxiety and producing sedation. When the ligand receptor site is boud to endogenous GABA, the GABA-A ion channel opens conducts Cl- ions through its pore. The Cl- influx causes hyperpolarization (internal membrane environment more negative than normal and comparatively more polarized (or negative) to the external membrane environment). Hyperpolarization prevents the likelihood of action potential generation and has an inhibitory effect on neurotransmission because Na+ dependant voltage gated channels cannot open unless the internal environment reaches threshold potential which is unlikely with the internal Cl- concentration greater than at resting potential.

GABA-B receptors are metabotropic, heterodimeric G-protein coupled receptors. GABA or exogenous agonist binding triggers a cascade of activity involving G-protein activation culminating in hyperpolarization of the neuron where the axonal environment is even more negatively charged that at normal resting potential. Hyperpolarization makes Na+ voltage gated channels highly unlikely to open (they open at threshold when the neuronal membrane potential difference approaches polarization) thus action potential firing is inhibited. GABA-B receptors are particularly important because they inhibit neurotransmitter release on pre-synaptic neurons.

G-protein activation can result in a hyperpolarized enrivornment in three ways. Firstly, activation of K+ channels during resting membrane potential (depolarization) means an efflux of K+ ions occurs down its concentration gradient leaving the internal environment more negative than normal due to the negatively charged protein content. This may have an effect anywhere on a neuron interupting conduction.
Inhibition of neurotransmitter release occurs by inhibiting Ca2+ influx into the pre-synaptic neuron and reducing its conductance within the neuron. The hyperpolarization caused by K+ channel opening and K+ efflux inhibits the opening of voltage gated calcium channels (VDCC). Ca2+ ions are necessary as they act as signalling molecules for exocytosis by stimulating Ca2+ sensitive proteins on the surface of vesicles. Also, inhibition of adenyl cyclase reduces coversion of ATP to cAMP which in turn reduces Ca2+ conductance in the neuron

Recap video of transmission at synapses:

Pathophysiology of anxiety disorders

People who suffer anxiety disorders may have persistant irrational fears producing ongoing severe symtoms or have less irrational fears that produce an anxiety response above what one would expect as normal physiological symptoms. Current understanding indicates the difference between normal and pathological anxiety is based around abnormalities in the modulation of anxiety/fear pathways resulting in increased stimulatory output from the amygdala to the hypothalamus.

Three neurochemicals may be involved:-

• Noradrenaline (locus coeruleus circuits) - excitatory
• Seratonin (raphe nuclei circuits) - inhibitory
• GABA (pathways involving the cortex) - inhibitory

Noradrenergic Model

• Locus coeruleus (nuclei situated in the brainstem) is involved in enhancing physiological responses to fear and panic
• Principal brain site for noradrenaline synthesis and location of origins for most noradrenergic neurons. Noradrenergic neurons can stimulate glutamate neurons in the brain.
• The LC has an excitatory effect on many brain circuits and structures including the cortex, amygdala, hippocampus and the hypothalamus, at the same time it can be activate my inputs from the precortex and amygdala.
• In response to a perceived threat, LC stimulation can in turn prime structures to produce an autonomic response via the hypothalamus.
• In some GAD patients alpha2 NA receptors have been shown to be down-regulated through chronic noradrenergic activity. Alpha2 receptors when agonized by noradrenalin and adrenalin are generally inhibitory producing vasodilation instead of vasoconstriction importantly produce negative feedback inbihiting the LC and further noradrenaline release within the brain.
• In some SAD patients, there is an hyperactive adrenocortical response to psychologic stress.
• In conclusion the ANS of many patients is hypersensitive and over-reactive to various for example anxiogenic drugs more or less specific to the LC (e.g. yohibine - alpha2 adrenergic receptor antagonist) stimulate LC firing increasing NA release stimulating glutamate release and neural anxiety circuits and can produce feelings of anxiety. In comparison anxiolytic drugs (e.g. benzodiazipines) can inhibit LC firing.

Noradrenaline a2 adrenergic receptor & NA neurons of the brain

There are two different types of adrenoreceptor – the α and β receptors. The α receptors are further classified into α1 and α2 subtypes and the β receptors are further classified into β1, β2 and β3 subtypes. The α2 adrenoreceptors are widely distributed throughout the body and are found in adrenergic neurones, blood vessels, the pancreas and in smooth muscle. Coupled to inhibitory G-proteins,α2 adrenoreceptors have an inhibitory effect on neurotransmission when bound by an agonist.

GABA Model

• GABA (Gamma amino butyric acid) is the primary inhibitory neurotransmitter in the CNS
• GABAergic neurons has an inhibitory effect when they synapse onto other neurons
• If there is a hypofunction in GABAergic transmission in the brain, activity in cortical regions may be overly excitatory and insufficient modulation of anxiety may occur. The hippocampus also has a notably high number of GABA receptors
• Benzodiazepines and other similar anxiolytic drugs bind to an allosteric site on the GABA-A receptor augmenting the inhibitory effect of GABA on other neurons, reducing feelings of anxiety and anxiety response.
• Some current hypotheses and findings for GABA-A receptor involvement include abnormal sensitivity of the allosteric benzodiazepine binding sites to antagonization and decreased receptor binding in panic disorder. In GAD, decreased binding with benzodiazepines occured in the left temporal lobe. The role of endogenous benzodiazepines is a current research area

Seratonin Model

• 5-HT is mostly an inhibitory neurotransmitter 
• The raphe nuclei (brainstem) is the major point of origin of seratogenic neurons. These neurons project widely throughout brain.
• It is thought that seratogenic neurons are important in inhibiting the locus coeruleus and diminishing noradrenergic output.
• Noradrenalin may have a direct effect inhibiting 5HT release from pre-synaptic terminals.
• Abnormalities may occur at 5HT(1A/1D) autoreceptor, with their upregulation/increased sensitivity inhibiting presynaptic seratonin production and release
• Abnormalities at SERT
• Abnormalities of expression of post synaptic 5-HT receptors (e.g. fewer 1A vs more 2A/2C receptors)- the agonization of the 5HT2A receptor in the limbic system results in avoidance and anxious responses.

Implicit versus explicit memory

Implicit memory involves memories encoded and stored based on what we have learned from past experience. It is used without conscious awareness and is non-verbalized or non-declarative. Step-by-step tasks such as brushing teeth, riding a bike and tying shoe laces are procedural memory examples calling on this unconscious memory, in other words we know how to do something we have done many times before, but because we are not consciously aware of its use, it is non-accessible and hard to verbalize. Forming memory stored as implicit initially can be difficult and is done by completing many repetitive trials, but equally it is very difficult to unlearn. Another category of implicit memory are our conditioned emotional responses where a conditioned stimulus familiar to us elicits a conditioned emotional response. In this way fear conditioned responses are based on implicit memory. We react in the same way to a aversive stimulus we have encountered before without conscious awareness. 

Explicit memory involves conscious recollection of objects, events, figures and faces. Episodic/Autobiographical memory allows us to remember what has happened in our lives. We use this type of memory to recall what happened at a certain event such as who got married to who on a certain date. It also allows us to remember and contextualize the details associated with the event. An example would not only be who married who, but where, what did you eat, who did you sit next to, what colour tie did you wear and so on. The quality of memorization depends on our state of mind and emotions at the time of the event and how often we reaccess these memories. Without repetition we may lose the ability to recall the memories or begin to recall memories with less accuracy or out of context, so one wedding may get confused with another.

Semantic memory concerns our knowledge of the external world. Although conscious it involves effortless, rapid retrieval. It includes the meaning of language and facts and figures. Unlike episodic memory, semantic memory does not include spacial/temporal significance or when or where we learned the facts, figures or meanings, it simply encodes fact and its relevance.

The role of the amygdala in fear

The amygdala has long been implicated in emotional learning in particular fear.

Removal of the amygdala from animals produces a fearless animal, yet for obvious ethical reasons, removal of the amygdala from humans hasn't been tried.

Recently a woman has been found without an amygdala. She has been threatened by a knife and held at knife point. Taking her to a petshop, she had no fear of snake of any size or venomousness and had to stopped by researchers from holding the tarantulas due to the risk of getting bitten. Fear was instead reproduced by a heightened state of arousal and level of interest.

Interestingly, when shown scary films she responded with the normal emotions, i.e. disgust at graphic scenes and laughter at comedy, but at no time reported or displayed symptoms of fear. It strengthens evidence that the amygdala is the neural seat of fear, but questions its role in other emotions.

BBC News - Woman who cannot feel fear may help in treating PTSD
The Human Amygdala and the Induction and Experience of Fear

The neurobiology of anxiety and fear

The amygdala is implicated in both anxiety and fear. The amygdala are a pair of almond shaped nuclei deep in the medial temporal lobes of the brain, considered part of the basal ganglia and along with the hippocampus, are considered the major components of the limbic system.
The amygdala function is the memory of emotional responses to past experiences. In memorising our emotional responses, when we encounter negative stimuli that we have come across in the past, the amygdala can elicit a more rapid, instinctive response as may be involved in fear. It is thought that our conditioned response to fear can take one of two routes. The long route involves the higher centers of the cerebral cortex and the hippocampus producing a more accurate response analysing whether the perceived threat is true or not. The short route is quick and 'dirty' as in it produces a rapid conditioned response to a perceived threat without analysis of whether the threat is real or not.

The long route - Slow and accurate
Sensory stimulus is processed and relayed by the nuclei of the thalamus. These include the lateral geniculate nuclei for visual stimulus, the medial geniculate nuclei for auditory stimulus and the ventral posterior nucleus involved in processing of proprioception, touch, temperature, pain, etc.


The normal response to sensory stimulus is the relay of information from the thalmic nuclei to the relevant cortical areas of the brain, such as the primary visual centre in the occipital lobe for vision and so on. These cortical centres appraise the stimulus for meaning and are involved in accurate targeting of the stimulus to the relevant brain structures. From the cortex, the information is relayed either directly to the amygdala or indirectly via the hippocampus.
The hippocampus is also part of the limbic system and is involved in explicit memory. It will contextualise the stimulus with past memories, for example where, when and with whom did the past experience occur. In response to new stimuli (as well as autobiographical memory in general) the hippocampus helps in memory formation of new events.
The amygdala integrates information about the appraised and contextualised stimulus as to whether the threat is real or not, before eliciting an emotion response via the autonomic nervous system.

Green arrow = Long route, Purple = Short route

The short route - Quick and dirty
The short route is thought to pass directly from the thalamic nuclei to the amygdala bypassing the higher centres and the hippocampus. The emotional response to stimulus is much faster and occurs as a fear conditioned response assuming the perceived stimulus is true. Higher centres are not involved so there is little discrimination of the threat as to whether it is true or not and only the characteristics of the threat are analysed. The longer route may occur simultaneously and be used to confirm whether the rapid response was correct or not. An example may be our initial response to a loud noise or bang to jump. The longer route will analyse whether the noise was really a threat or not and from what direction it came.

Fear vs Anxiety - are they the same?

Fear n. an emotional state evoked by the threat of danger and usually characterized by unpleasant subjective experiences as well as physiological and behavioural changes. Fear is often distinguished from anxiety in having a specific object. Associated physiological changes include increases in heart rate, blood pressure, sweating, etc. (Oxford Medical Dictionary)
Fear n. an unpleasant often strong emotion caused by anticipation or awareness of danger and accompanied by increased autonomic activity (Medline Plus)

Anxiety n. a generalized pervasive fear. (Oxford Medical Dictionary)
Anxiety n. 1: a painful or apprehensive uneasiness of mind usually over an impending or anticipated ill 
2: an abnormal and overwhelming sense of apprehension and fear often marked by physiological signs (as sweating, tension, and increased pulse), by doubt concerning the reality and nature of the threat, and by self-doubt about one's capacity to cope with it. (Medline Plus)

From the above definitions fear and anxiety have much in common and in common language fear and anxiety may be used synonymously. Fear is an emotional response whilst anxiety is a psychological and physiological state and both are unpleasant, yet despite this unpleasantness both can be considered natural responses with evolutionary roles. Both have roles in protecting ourselves or others from negative consequences by eliciting appropriate reactions in difficult situations.

Fear is more specific and usually driven by more external stimuli. The impeding threat may be well defined as may the consequences. An example may be an oncoming car speeding towards us. The natural reaction is quick one of escape and avoidance, to get out of the way of the oncoming car. An evolutionary example would be of that of encountering a lion or snake inducing fear producing a survival driven response to flee the situation.

Fear conditioning is the learned recognition and appropriate response of an aversive stimulus and fears are largely conditioned human behaviour. The recognition of a stimulus as aversive may be learned from personal experience of bad outcomes such as a child learning of the hazards of water by near-drowning, or through observing the outcomes of others. Many fears are learned through experience in early childhood because babies naturally fear few things. Loud noises are one example of unconditioned fears related to sensory overload that produce an unconditioned response (usually crying) in babies. In Watson's Little Albert experiment, Albert wasn't frightened of the white rat. He was however upset by loud noise, but wasn't able to predict when loud noise might occur. The rat was a neutral stimulus, he had no fear conditioned or unconditioned towards the rat, in fact he appeared to like the white rat. The white rat (neutral stimulus) was then paired with the loud sound (unconditioned stimulus) producing the unconditioned response of crying. Over time the introduction of the white rat would make Albert cry, thus the rat had become a conditioned stimulus and the anticipatory fear it produced was the conditioned response, even if no loud sound occurred. Fear generalization also occurred where Little Albert associated anything fluffy and white as being a condtioned fear stimulus associated with the noise outcome.

Anxiety can be perceived as being different to fear in that its stimulus is less specific and often less externalized and may be related to scenarios that are less controllable or avoidable. Unlike fear which is a conditioned negative emotional response, anxiety may not have an identifiable trigger as its stimulus and produces a state of mind with generalized fear and negative emotion, underlying heightened arousal and perception of sensory inputs. Anxiety can also be seen as a future-oriented state of mind versus fear which may be a subjective emotion related to a stimulus being perceived at the present. Anxiety as a normal physiological state may serve to prepare us or keep us in a heightened state of arousal to interpret and cope with new or impending threats and to learn and adapt new and suitable responsive behaviours. If however the level of anxiety is disproportionate or occurs non-objectively to stimuli or situations not normally deemed as threatening or dangerous it may be considered unhelpful and possibly pathological if it results it changes to the ability to function normally or starts to induce abnormal learned behaviours such as avoidance or ongoing generalized perpetual fear.